Interlaboratory transfer of a PCR multiplex method for simultaneous detection of four genetically modified maize lines: Bt11, MON810, T25, and GA21

The number of cultured hectares and commercialized genetically modified organisms (GMOs) has increased exponentially in the past 9 years. Governments in many countries have established a policy of labeling all food and feed containing or produced by GMOs. Consequently, versatile, laboratory-transferable GMO detection methods are in increasing demand. Here, we describe a qualitative PCR-based multiplex method for simultaneous detection and identification of four genetically modified maize lines: Bt11, MON810, T25, and GA21. The described system is based on the use of five primers directed to specific sequences in these insertion events. Primers were used in a single optimized multiplex PCR reaction, and sequences of the amplified fragments are reported. The assay allows amplification of the MON810 event from the 35S promoter to the hsp intron yielding a 468 bp amplicon. Amplification of the Bt11 and T25 events from the 35S promoter to the PAT gene yielded two different amplicons of 280 and 177 bp, respectively, whereas amplification of the 5' flanking region of the GA21 gave rise to an amplicon of 72 bp. These fragments are clearly distinguishable in agarose gels and have been reproduced successfully in a different laboratory. Hence, the proposed method comprises a rapid, simple, reliable, and sensitive (down to 0.05%) PCR-based assay, suitable for detection of these four GM maize lines in a single reaction.     

The role of lipoprotein lipase (LPL) in the incorporation of neutral lipids into the oocytes of the European sea bass (Dicentrarchus labrax L.) during gonadal development

We have cloned the cDNA encoding LPL and studied its mRNA expression and enzyme activity in the ovary of European sea bass in order to investigate a possible role of this enzyme in the incorporation of neutral lipids into the oocytes. The results suggest that LPL is likely to play an important role in this process.     

A simple field method for assessing the ecological quality of riparian habitat in rivers and streams: QBR index

• 1. An index of riparian quality useful for the management of streams and rivers is presented. The purpose of the index is to provide managers with a simple method to evaluate riparian habitat quality. The index is easy to calculate and can be used together with any other index of water quality to assess the ecological status of streams and rivers. It may also be a useful tool for defining ‘high ecological status’ under the EC Water Framework Directive.
• 2. The index, named QBR, is based on four components of riparian habitat: total riparian vegetation cover, cover structure, cover quality and channel alterations. It also takes into account differences in the geomorphology of the river from its headwaters to the lower reaches. These differences are measured in a simple, quantitative way. The index score varies between 0 and 100 points.
• 3. The QBR index is calculated in the field through a two‐sided A4 page form that may be completed in 10 min.
• 4. The development of the QBR index included trials in four Mediterranean stream catchments in Catalonia (NE Spain). Seventy‐two sampling sites were assessed and results were used to test the index.
• 5. No taxonomic expertise is needed to apply the index, although some knowledge of local flora is required to differentiate between native and non‐native tree species.
• 6. These results show that the QBR index may be used despite regional differences in plant communities. The quality ranges obtained when the index is applied are not heavily influenced by observers at the same site.
• 7. At present, the index is being used by different research teams and tested in a comparative study of 12 watersheds along the Mediterranean Spanish coast.
• 8. It is expected that the QBR index may be adapted for use in other geographical areas in temperate and semi‐arid zones without changes in the index rationale.

Copyright © 2002 John Wiley & Sons, Ltd.     

Determination of the Bioavailable Fraction of Cu and Zn in Soils Amended with Biosolids as Determined by Diffusive Gradients in Thin Films (DGT), BCR Sequential Extraction, and Ryegrass Plant

This study assessed the effect of biosolids applied at rates, 0, 30, 45, and 60?Mg?ha^?1 on the chemical associations and bioavailability of Cu and Zn in soils from an important agricultural zone of the Metropolitan Region in Central Chile. Three methods were used to determine the bioavailability of Cu and Zn in soils: ryegrass (Lolium perenne) plants, diffusive gradients in thin films (DGT) technique, and Community Bureau of Reference (BCR) sequential extraction. The DGT effective concentration (C _E) and sequential extract acid soluble fraction of the BCR extraction (most labile fraction of the soils, normally associated with bioavailability) were compared with total metal concentration in ryegrass plants as a means to compare the chemical and biological measures of bioavailability. Total Zn was higher in comparison to Cu for all treatments. Concentrations were within the limits set by the Chilean regulations for land-applied biosolids. Metals in the control soil were primarily found in the residual fraction of soils. Biosolids application generally decreased this fraction, with a subsequent increase observed mainly in the acid soluble fraction. The contents of Cu and Zn in ryegrass plants increased with increasing rates of biosolids. Comparison of the Cu and Zn content in ryegrass plants with C _E, showed a good correlation for Zn. However, the C _E for soil Cu was only related to plant Cu for some of the soils studied. Correlation between Zn in ryegrass plants and the labile fraction of Zn as measured by the sequential extraction was excellent, with correlation coefficients >0.9, while for Cu, correlation coefficients were lower.     

Baseflow control on sediment flux connectivity: Insights from a nested catchment study in Central Mexico

In order to assess the extent of sediment connectivity between uplands and lowlands and to quantify the processes of in-channel deposition and remobilization, measurements of suspended sediment fluxes were conducted in a nested rural catchment of the Mexican Volcanic Belt. Data were collected over one year at three upland sites (3 to 12 km²) and two downstream stations (390–630 km²). Our results show that a structural discontinuity in the catchment (i.e. abrupt slope decrease at the junction between piedmonts and the alluvial plain from 2 to 10% to < 0.1%) could be compensated by functional continuity during floods. Direct conveyance of fine sediment to the outlet occurred when a high stream transport capacity was reached. Erosion of the streambed was observed on various occasions and accounted for up to 50% of the flux leaving the catchment during one event. Conversely, temporary in-channel storage was apparent on other occasions, amounting to up to 52% of the flux recorded upstream during one storm. These two distinct behaviours were approximately equally distributed along the rainy season and strongly driven by the extent of coupling between surface and subsurface water. This work indeed highlights the role of baseflow spatial variations in determining the extent of lowland sediment conveyance. Riverbed erosional processes occurred when large differences in pre-event baseflow values (i.e. at least a twofold longitudinal increase) were observed between the 5-km distant lowland stations. Our findings outline the importance of systematically taking into consideration the baseflow parameter in research focusing on fine sediment transport across scales.     

Effect of biosolid application to Mollisol Chilean soils on the bioavailability of heavy metals (Cu,Cr, Ni,and Zn) as assessed by bioassays with sunflower (Helianthus annuus) and DGT measurements

Purpose

This study assessed the effect of biosolid application on the bioavailable fraction of some trace elements (Cu, Cr, Ni, and Zn) using a bioassay with sunflower (Helianthus annuus) and a chemical assay, diffusion gradient in thin films (DGT).

Materials and methods

Five surface soil samples (0–20 cm) were collected from an agricultural zone in Central Chile where biosolids are likely to be applied. Municipal biosolids were mixed with the soil at concentrations of 0, 30, 90, and 200 Mg ha^?1. The experiment to determine the bioavailability of metals in the soil using the bioassay was performed using sunflower. The DGT technique and Community Bureau of Reference (BCR) sequential extraction were used to determine the bioavailable fractions of the metals.

Results and discussion

The application of biosolids increased the phytoavailability of Zn, Ni, and Cr in most of the soils, as indicated by the increasing concentrations in sunflower plants as the biosolid application rate increased. In two of the soils, Codigua and Pelvín, this increase peaked at an application rate of 90 Mg ha^?1. Decreases in the bioavailable fractions of Zn, Ni, and Cr were observed with higher biosolid application rates. The bioavailability of metals was estimated through multiple linear regression models between the metals in the sunflower plants and the different chemical fractions of metals in the soils treated with different biosolid rates, which displayed a positive contribution of the labile (water soluble, carbonate, and exchangeable), oxide, and organic metal forms in the soil, particularly with respect to Ni and Zn at application rates of 30 and 90 Mg ha^?1. The bioavailable fraction of metals was determined in soils using the DGT technique. The effective concentration (C _E) results were compared with those in sunflower plants. The DGT technique could effectively predict the bioavailable fractions of Cr, Ni, and Zn in the Taqueral soil but only that of Zn in the Polpaico soil.

Conclusions

The application of biosolids significantly increased the labile fraction of most of the metals in the studied soils, particularly at the highest biosolid application rate. C _E increased as the concentration of biosolids increased for most of the metals. The effectiveness of the DGT technique for predicting the bioavailability of metals was dependent on the soil type and the metal. However, the C _E for soil Cu was not related to plant Cu for all soils studied.     

Different sensitivity of isoprene emission, respiration and photosynthesis to high growth temperature coupled with drought stress in black poplar (Populus nigra) saplings

The effects of the interaction between high growth temperatures and water stress on gas-exchange properties of Populus nigra saplings were investigated. Water stress was expressed as a function of soil water content (SWC) or fraction of transpirable soil water (FTSW). Isoprene emission and photosynthesis (A) did not acclimate in response to elevated temperature, whereas dark (R(n)) and light (R(d)) respiration underwent thermal acclimation. R(d) was ~30% lower than R(n) irrespective of growth temperature and water stress level. Water stress induced a sharp decline, but not a complete inhibition, of both R(n) and R(d). There was no significant effect of high growth temperature on the responses of A, stomatal conductance (g(s)), isoprene emission, R(n) or R(d) to FTSW. High growth temperature resulted in a significant increase in the SWC endpoint. Photosynthesis was limited mainly by CO(2) acquisition in water-stressed plants. Impaired carbon metabolism became apparent only at the FTSW endpoint. Photosynthesis was restored in about a week following rewatering, indicating transient biochemical limitations. The kinetics of isoprene emission in response to FTSW confirmed that water stress uncouples the emission of isoprene from A, isoprene emission being unaffected by decreasing g(s). The different kinetics of A, respiration and isoprene emission in response to the interaction between high temperature and water stress led to rising R(d)/A ratio and amount of carbon lost as isoprene. Since respiration and isoprene sensitivity are much lower than A sensitivity to water stress, temperature interactions with water stress may dominate poplar acclimatory capability and maintenance of carbon homeostasis under climate change scenarios. Furthermore, predicted temperature increases in arid environments may reduce the amount of soil water that can be extracted before plant gas exchange decreases, exacerbating the effects of water stress even if soil water availability is not directly affected.